Blood coagulation is a complex process involving many activating and inactivating coagulation factors. Anticoagulant proteins are known to be important for regulation of the coagulation process. See B. Lammle and J. Griffin (Clinics in Haemotolog 14, p. 281-342, 1985) for a review on coagulation inhibitors and regulation of coagulation.
Thus heparin is used clinically to increase the activity of antithrombin III and heparin cofactor II. Antithrombin III is used for the inhibition of factor Xa and thrombin. Hirudin is used for the inhibition of thrombin Protein C may be used for the inhibition of factors V and VIII.
Coagulation can be initiated through the extrinsic pathway by the release of tissue factor (J.H. Morrissey et al.: Thromb Res 50, p. 481-93, 1988). Coagulation activation by the extrinsic pathway may be inhibited by different mechanisms (P.M. Sandset et al.: Thromb Res 47, p. 389-400, 1987; B.J. Warn-Cramer et al.: Thromb Res 4s, p. 11-22, 1987; S. Kondo and W. Kisiel: Blood 70, p. 1947-54, 1987; S.D. Carson: J. Biol Chem 262, p. 718-21, 1987; G.J. Broze et al.: Blood 77, p. 335-43, 1988; S. Kondo et al.: Thromb Res 4s, p. 449-59, 1987).
The basic trigger in many coagulation disorders is the release of tissue factor and thus activation of factor X by factor VII-tissue factor. During surgery, tissue factor is released and thrombi may be formed In heart attack a primary thrombus is formed and when this thrombus is released, tissue factor is exposed and coagulation is initiated resulting in a secondary, perhaps lethal thrombus. During sepsis, bacterial endotoxin induces the systemic release of tissue factor. This may lead to disseminated intravascular coagulation (DIC). DIC can be treated with antithrombin III (T.E. Emerson et al.: Circulatory Shock 21, p. 1-13, 1987) Which inhibits the late steps in the coagulation cascade. Activated Protein C which inhibits in the middle of the coagulation cascade can also be used for the treatment of DIC (F.B. Taylor et al.: J Clin Invest 79, p. 918-25, 1987).
Protein showing extrinsic coagulation Pathway Inhibitor (EPI) activity has been recovered and isolated from human cells. It is known that EPI inhibits factor VII-tissue factor catalyzed activation of FX. However, the exact mechanism by which EPI inhibits coagulation is not known. Human plasma contains 3 molecular species showing EPI activity. The molecular masses are&gt;500 kDa, 200 kDa and 40 kDa respectively (P.M. Sandset et al.: Thromb Res 47, p. 389-400, 1987).
The object of the present invention is an improved method to isolate the protein EPI in concentrated or pure form.